Binding of small molecular ligands to proteins plays an essential role in a large number of biological and chemical processes. Molecular recognition of a ligand in its protein binding pocket has long been a subject of great importance for understanding cellular functions of proteins in biology. Here, the term “molecular recognition” refers to the affinity and specificity of ligand binding to its targeted protein, which is governed by non-bonded intermolecular interactions. Thus, quantum chemical analysis of non-bonded intermolecular interactions has emerged as one of the frontiers of theoretical research. Historically, non-bonded interactions have been extensively studied for small prototype molecular systems by high-level theoretical calculations. However, applications of high-level quantum chemical treatments to large biomolecular systems are limited by the availability of computing power until now. Thus, this work on quantum chemical analysis of protein-ligand interactions in several biochemical systems represents a timely undertaking.
Human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) is a major target for the development of anti-HIV drugs, and more than half of the currently approved anti-AIDS drugs target HIV-1 RT. Non-nucleoside reverse transcriptase inhibitors (NNRTIs) represent a major class of such drugs. However, one of the severe drawbacks for NNRTI is that its efficacy for anti-AIDS therapy is seriously impaired by the rapid emergence of drug-resistant mutants. In particular, it has been reported that mutation of aromatic residues to non-aromatic residues, such as Y181C and Y188C, resulted in severe drug resistance to the first generation NNRTIs like nevirapine. On the other hand, second generation NNRTIs like efavirenz are more resilient to mutations at Tyr181 and Tyr188. A large scale data mining and high level quantum chemical analysis was performed to investigate the intermolecular interactions governing molecular recognition between Non-Nucleoside Reverse Transcriptase Inhibitors (NNRTIs) and HIV-1 RT. A total of 66 non-redundant X-ray crystal structures of HIV-1 RT with bound NNRTI were extracted from the Protein Data Bank, among which 11 structures of HIV-1 RT in complexes with nevirapine and 5 structures of HIV-1 RT in complexes with efavirenz have been chosen. By analyzing the alignment of all available 3D structures of HIV-1 RT in complexes with NNRTIs through superimposition of nevirapine or efavirenz, it was found that CH-p and p-p stacking interactions are employed by HIV-1 RT for recognition of NNRTIs. Subsequently, the intermolecular interaction energies between NNRTIs and their surrounding residues were calculated by ab initio electronic structure calculations at the MP2/6-311++G** level using the supermolecular approach, coupled with solvation energy correction through the SM5.42R Solvation Model. As a significant outcome of this project, the molecular determinants responsible for the differential response of the first generation NNRTI nevirapine (NEV) and second generation NNRTI efavirenz (EFZ) to the primary mutation of HIV-1 RT are unraveled. ab initio electronic structure calculations revealed that the binding of the first generation NNRTI nevirapine to its protein pocket is dominantly achieved through p-p stacking interactions between the aromatic moiety of nevirapine and aromatic residues, whereas CH-p interactions are more important than p-p stacking interactions for efavirenz binding to wild-type RT. Since p-p stacking interactions are less important for the binding of the second generation NNRTI efavirenz, mutation of aromatic residues Tyr181 and Tyr188 do not lead to significant loss of binding affinity. That explains why the second generation NNRTIs exhibit better drug resistance profiles to single point mutation of the HIV-1 RT. In addition, conformational flexibility of efavirenz may also partially account for its potency against single point mutations. This work not only yielded an in-depth understanding of the molecular recognition of NNRTIs in HIV-1 RT, but also provided molecular level insights into designing new generation NNRTI drugs that are less sensitive to drug resistance mutations.
Adenosine 5'-triphosphate (ATP) plays a pivotal role in all forms of life. The second project aims at understanding molecular recognition of the adenine moiety of ATP in ATP-binding proteins. A large scale data mining of the Protein Data Bank was conducted to study the role of CH-p interactions in binding of the adenine base. Alignment of 68 high-resolution, non-redundant three-dimensional crystal structures of adenine-protein complexes obtained from the Protein Data Bank shows that in 41 out of 68 (60%) adenine-protein complexes, CH-p interactions between the aromatic ring of adenine and aliphatic amino acid residues do exist. The majority of aliphatic residues tend to cluster on the top and bottom of the aromatic p ring of the adenine base with the CH group pointing directly toward the face of the aromatic ring. Subsequently, the intermolecular CH-p interaction energies between adenine bases and their interacting protein residues were calculated by ab initio electronic structure calculations at the MP2/6-311++G** level using the supermolecular approach. Among all the representative CH-p interaction pairs studied, it was found that the strength of CH-p interactions ranges from -0.05 kcal/mol to -2.28 kcal/mol at the MP2 level, varying with intermolecular configurations, i.e., interacting distance and orientation. This study of CH-p interactions in ATP-binding proteins complements a previous study done in the Hu lab in which three other modes of non-bonded intermolecular interactions for binding ATP were investigated. Together, the two studies yielded a complete picture for molecular recognition of the adenine base in ATP-binding proteins. On average, the number of non-bonded interactions between the adenine base and protein in each adenine-protein complex is 4.0 aliphatic residues for CH-p interactions, 2.7 hydrogen bonding interactions, 1.0 p-p stacking interaction, 0.8 cation-p interactions. Although the strength of CH-p interactions is generally weak due to its physical nature of dispersion interaction, the importance of CH-p interactions for binding of the adenine base in ATP-binding proteins should not be underestimated. This is consistent with expectation, our calculations have shown that the energetic cost of dehydration is much lower for the CH-p interactions in comparison with hydrogen bonding and cation-p interactions.
FK506 (tacrolimus) and rapamycin (sirolimus) are natural products that are used as immunosuppressive drugs to prevent rejection in organ transplantation. The drugs function with a unique mode of action. Prior to binding to their protein targets, these drugs form a complex with an endogenous chaperone FK506-binding protein 12 (FKBP12). The resulting binary complexes of FKBP-FK506 and FKBP-rapamycin inhibit the target protein calcineurin and mTOR, leading to immunosuppressive activity. The crystal structures reveal that the binary complexes bind to the target proteins with high affinity and specificity on relatively large and flat surfaces that are considered “undruggable” to conventional small molecules. Exploring this unique mode of drug action, our collaborators Pei and Briesewitzat at the Ohio State University attempted a novel approach for designing new drugs. It was hypothesized that it may be possible to extend the mechanism of action of FK506 and rapamycin to target other undruggable protein sites. To test the hypothesis, our collaborators have developed a synthetic route to synthesize a large library of cyclic bifunctional molecules. These synthetic molecules comprise a common FKBP12-binding moiety but different effector domains, and are called rapalogs. It is hoped that these rapalogs may create diverse composite binding surfaces on FKBP12, which may be screened for binding to other protein targets. The designed rapalogs were tested for binding to FKBP12 by a fluorescence polarization competition assay. It was found that majority of the rapalogs (163 compounds) bound to FKBP12 with excellent to respectable affinities. In particular, two major findings are achieved: (1) rapalogs with Asp at the R3 position bound to FKBP12 more potently, whereas those with Lys at the R3 position were generally less effective ligands; and (2) the residue at the R4 position had negligible effect on the binding affinity, except for Asp, which yielded high activities even when the R3 residue was not optimal. In order to understand the experimental observations at the molecular level, we performed a hybrid quantum mechanical/molecular mechanical (QM/MM) molecular modeling to optimize the geometries of the complexes of FKBP12 with two potent rapalogs. The FKBP12-rapalog complexes were modeled with a two stage protocol. At the first stage, a three dimensional structure for the rapalog was generated by geometry optimization with the SPARTAN 02 program. At the second stage, the optimized rapalog was docked into FKBP12 by means of hybrid QM (quantum mechanics)/MM (molecular mechanics) optimization to form the FKBP12-rapalog complex. The QM layer contains rapalog and its direct interacting residues and is treated at the PM3 level. The MM layer includes the rest of the entire FKBP12 protein and is described by the AMBER force field. The optimized FKBP12-rapalog complexes provide molecular level insights into binding of each rapalog with FKBP12. A key FKBP-rapalog binding interaction occurs in the form of CH-p interactions between the side chain of the FKBP-binding motif and the hydrophobic pocket formed by the side chains of aromatic residues Tyr26, Phe46, Trp59, and Phe99 of FKBP12. The carbonyl groups of the FKBP-binding motif also form two key hydrogen bonds with the side chain of Tyr82 and the main chain amide-NH of Ile56, respectively. In particular, the side chain of Asp at position R3 is physically proximal to the guanidinium group of Arg42 of FKBP12 and forms a favorable electrostatic interaction. In contrast, a Lys residue at the same position introduces an unfavorable electrostatic interaction with Arg42. The above results are in excellent agreement with the experimental observations that rapalogs with Asp at the R3 position bound to FKBP12 more potently, whereas those with Lys at the R3 position were generally less effective ligands.